Interkinetic Nuclear Migration Is a Broadly Conserved Feature of Cell Division in Pseudostratified Epithelia  Emily J. Meyer, Aissam Ikmi, Matthew C. Gibson  Current Biology  Volume 21, Issue 6, Pages 485-491 (March 2011) DOI: 10.1016/j.cub.2011.02.002 Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 1 Interkinetic Nuclear Migration-like Mitotic Cell Behavior in the Drosophila Wing Disc (A) neuroglian-GFP;histone H2-RFP third-instar wing imaginal disc. (B) Anti-phosphohistone H3 (PH3)+ mitotic figures (blue) colocalize with the plane of the septate junctions as labeled by Discs large (DLG, green). Phalloidin staining of F-actin (ACT) is red. Terminal stages of cytokinetic furrow contraction are visible as F-actin-rich foci, also confined to the plane of the septate junctions (white arrows). (C) Stages of mitosis (interphase, prophase, metaphase, anaphase, telophase, and cytokinesis) from fixed samples labeled for DLG (green), mitotic chromatin (blue), and F-actin (red). Note the continuous localization of mitotic figures to the plane of the septate junctions. (D) Phalloidin-stained cross-section through a wing imaginal disc. Yellow arrows indicate rounded and presumably mitotic cells at the apical epithelial surface. (E) Stochastically labeled GFP+ clones (green) illustrate the variability in nuclear positioning (white asterisks) in interphase cells. (F) Schematic representation of interphase nuclei relative to the polarized cell-cell junctions (adherens junctions, red; septate junctions, green; nuclei, blue; Ap, apical; Ba, basal). (G) Whereas interphase nuclei occupy medial positions within the ∼50 μm-thick epithelium (white asterisks), rounded cells and anti-PH3+ mitotic figures (blue) are restricted to the DLG-delimited mitotic zone (MZ, green). (H) In fixed discs stained as above, intense basal F-actin accumulation is first observed in prophase cells (leftmost panel, yellow arrow) and persists throughout mitosis. (I) In discs stained for anti-DLG (blue) and F-actin (red), anti-p-MRLC (green) accumulates at the cortex of early prophase figures and persists throughout mitotic rounding. Notably, p-MRLC is not detected in the F-actin-rich basal process (white arrows). Uniform cortical p-MRLC is lost or redistributed during formation and progression of the contractile ring (green arrowheads). Current Biology 2011 21, 485-491DOI: (10.1016/j.cub.2011.02.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 2 Rho Kinase and Cortical Contractility Are Required for Nuclear Translocation to the Mitotic Zone (A) Experimental design for cytoskeletal inhibitor studies. (B) Latrunculin A (LatA) treatment caused anti-PH3+ nuclei to accumulate basal to the MZ (yellow arrows). (C) Distance of 200 anti-PH3+ nuclei from the MZ for control discs (uncultured and 30 min of culture) compared with discs treated for 30 min with 100, 250, and 500 μM LatA. (D) Percentages of anti-PH3+ nuclei outside the MZ following LatA treatment (n = 300 nuclei per condition). (E and F) CytoD treatment (at 5, 50, and 100 μM) disrupted apical translocation of mitotic nuclei in a manner similar to LatA. (G) Percentage of PH3+ nuclei outside the MZ for CytoD treatments (n = 300 nuclei per condition). (H and I) Effects of Y-27632 (at 1, 2.5, and 5 mM) on the positions of 200 anti-PH3+ nuclei. (J) Percentage of PH3+ nuclei outside the MZ following treatment with Y-27632 (n = 300 nuclei per condition). Statistical analyses were performed with the Mantel-Haenszel test using controls cultured for 30 min. (K–N) Third-instar wing imaginal discs stained for phalloidin (F-actin), anti-p-MRLC, and anti-PH3. (K) UAS-dicer-2;nubbin-Gal4 control disc showing rounded mitotic cells specifically labeled with anti-p-MRLC. Box in main image indicates position of inset. (L) nubbin-Gal4>rokRNAi wing discs display a severe reduction of anti-p-MRLC staining as well as a reduction in the number of apically rounded cells within the nubbin-Gal4 expression domain (dotted line). Note that mitotic cells outside the nubbin-Gal4 domain exhibit normal anti-p-MRLC staining (white arrows). (M) xz section of a control disc showing cortical anti-p-MRLC staining in PH3+ cells. (N) In nubbin-Gal4>rokRNAi discs, cortical anti-p-MRLC is eliminated in PH3+ cells (white arrows). (O) UAS-dicer-2;nubbin-Gal4 control disc. (P) nubbin-Gal4>rokRNAi disc showing numerous anti-PH3+ nuclei (blue) basal to DLG accumulation at the septate junction (green). (Q) Percentage of PH3+ nuclei out of the MZ in nubbin-Gal4>rokRNAi discs (n = 450 nuclei per condition). Statistical analysis was performed using Student's t test. ∗p < 0.0001; ∗∗p < 0.00001. Error bars indicate standard deviation. Current Biology 2011 21, 485-491DOI: (10.1016/j.cub.2011.02.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 3 Microtubule Dynamics during Mitosis in the Drosophila Wing Disc (A) Apices of fixed wing disc cells exhibit a diffuse microtubule web during interphase (white asterisks). As cells enter prophase and begin to round up, the apical microtubule web disappears (white arrow). The mitotic spindle begins to form in subsequent stages (white arrowhead). (B–H) In xz sections through fixed discs, apical microtubules orient along the apicobasal axis. Apical anti-tubulin staining (green) diminishes in early prophase (B) and vanishes altogether as the anti-PH3+ nucleus (blue) reaches the MZ (C). After formation of the mitotic spindle (D and E) and cytokinesis, the midbody microtubules turn basally, ultimately becoming oriented along the apicobasal axis of the daughter cells (F–H). (I–K) Paclitaxel treatments were sufficient to cause a mitotic arrest (Table S3) but did not strongly affect the apicobasal positions of mitotic nuclei (yellow arrows in I) or mitotic cell rounding. (J) Plotted positions of 200 nuclei relative to the MZ for 50, 200, and 500 μM paclitaxel. (K) Percentages of anti-PH3+ nuclei basal to the MZ following paclitaxel treatments (n = 300 nuclei per condition). Statistical analyses in (K) and (N) were performed with the Mantel-Haenszel test using controls cultured for 30 min. ∗∗p < 0.0001. Error bars indicate standard deviation. (L–N) Colchicine treatment (at 1, 50, and 500 μM) caused a mitotic arrest (Table S3) but did not strongly affect the apicobasal positions of mitotic nuclei or mitotic cell rounding (yellow arrows in L). Current Biology 2011 21, 485-491DOI: (10.1016/j.cub.2011.02.002) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 4 Interkinetic Nuclear Migration in the Ectoderm of Nematostella vectensis (A) Differential interference contrast image of a 4-day-old planula larvae showing ectoderm (ect), endoderm (end), blastopore/oral pole (asterisk), and pharynx (pha). Scale bars represent 50 μm in (A)–(C). (B) Nematostella larvae stained with phalloidin to label F-actin (ACT, green) and propidium iodide to label nuclei (PI, red). (C) As in Drosophila (e.g., Figure 1B), the apical surface of the planular ectoderm exhibits rounded cells with apically localized nuclei. (D) Transverse view showing apical rounding of cells (white arrowheads). Scale bars represent 25 μm in (D) and (E). (E–F′) Detailed view of the ectoderm. Rounded cells in the MZ exhibit enriched cortical F-actin (white arrowheads). (G) Apical localization of anti-PH3+ nuclei (green) in the ectoderm of control larvae stained with anti-α-tub (red) and PI (blue). Scale bar represents 10 μm. (H and I) CytoD-treated (H) and colchicine-treated (I) animals exhibit anti-PH3+ nuclei in abnormal positions basal to the MZ. (J) Effects of CytoD and colchicine on the position of anti-PH3+ nuclei. Statistical analyses were performed with the Mantel-Haenszel test using controls cultured in dimethyl sulfoxide. ∗p < 0.0001; ∗∗p < 0.00001. Error bars indicate standard deviation. Current Biology 2011 21, 485-491DOI: (10.1016/j.cub.2011.02.002) Copyright © 2011 Elsevier Ltd Terms and Conditions